The top layer of human skin or the epidermis is composed of many different cell types including keratinocytes, melanocytes and langerhans cells. Keratinocytes are the major cell type of the epidermis (75-80% of the total number of cells in the human epidermis). Within the epidermis the keratinocytes reside in four distinct stages of differentiation. The basal layer rests on the basal lamina separating epidermis from the dermis. These cells are large columnar rapidly proliferating cells. These basal cells migrate upward within the epidermis, initiated by the process of differentiation. The layer above the basal cells is the spinous layer. The cells in the spinous layer initiate the production of proteins characteristic of the differentiated epidermis. The granular layer, lying above the spinous layer, is characterized by electron-dense granules. This layer is responsible for the synthesis of lipid molecules required for the formation of the water impermeable barrier of the skin. The topmost layer of the skin, the stratum corneum, is formed from the granular layer by the destruction of cellular organelles. The cells in the stratum corneum, corneocytes, contain extensively cross-linked proteins, surrounded by a highly resistant cell envelope. The corneocytes are embedded in a bed of specific lipid structures (analogous to bricks on a bed of mortar) and this structure provides the protective barrier for the skin. The outermost layer of corneocytes is peeled off from the skin during the normal process of desquamation. Differentiation of the epidermal keratinocytes is the driving force for the normal desquamation process to occur. Epidermal differentiation is important for providing the essential function of the skin, namely to provide a protective barrier against the outside environment and to prevent loss of water from the body. The basal cells which have the highest rate of growth, are the least differentiated. The most differentiated cells of the stratum corneum do not have the ability to grow.
Initiation of differentiation of keratinocytes is accompanied by inhibition of their growth. The rate of synthesis of DNA determined by the incorporation of radiolabeled substrate [.sup.3 H] thymidine, is an indicator of the rate of growth of the cells. A decrease in DNA synthesis therefore indicates decrease in growth and increase in differentiation of keratinocytes.
The present invention is based, in part, on the discovery that a combination of two specific active ingredients, namely 25-hydroxycholecaliciferol and short chain lipids, results in synergistic increase in differentiation, which in turn results in increased benefits to skin, such as improved conditioning, improved youthful appearance, decrease in wrinkle appearance, moisturizing, and treatment of photodamaged skin and various skin disorders.
Vitamin D.sub.3 is produced in the skin of mammals as the result of irradiation which converts 7-dehydrocholesterol into vitamin D.sub.3 in the presence of sufficient sunshine. Vitamin D.sub.3 is then metabolized into active biological metabolites. The majority of vitamin D.sub.3 is taken up by liver where it is hydroxylated at C-25. The resulting 25-hydroxycholecalciferol (hereinafter "25-OH-D.sub.3 ") is then transported to various target organs where further hydroxylation takes place at C-1 or C-24. Several published studies establish that skin is one of the organs in which synthesis of 1,25-dihydroxycholecalciferol (hereinafter "1,25-(OH).sub.2 D.sub.3 ") and 24,25-dihydroxycholecalciferol from 25-OH-D.sub.3 occurs. See e.g., Bikle et al., "1,25-Dihydroxyvitamin D.sub.3 Production by Human Keratinocytes", The Journal of Clinical Investigation, Inc., Volume 78, (August 1986), pp. 557-566.
1,25-(OH).sub.2 D.sub.3 is the major biologically active metabolite of vitamin D.sub.3. 1,25-(OH).sub.2 D.sub.3 plays a central role in regulating blood calcium levels by increasing bone resorption and calcium absorption from intestine. Recent studies indicate that exogenous or endogenous 1,25-(OH).sub.2 D.sub.3 inhibits DNA synthesis (i.e., inhibits growth) and induces differentiation of keratinocytes. See e.g., Pillai et al. "1,25-Dihydroxyvitamin D Production and Receptor Binding in Human Keratinocytes Varies with Differentiation" The Journal of Biological Chemistry, Vol. 263, No. 11, (Apr. 15, 1988), pp. 5390-95; and Hashimoto et al., "Growth-inhibitory effects of 1,25-Dihydroxyvitamin D.sub.3 on Normal and Psoriatic Keratinocytes" British Journal of Dermatology (1990) Vol. 123, pp. 93-98. Topical compositions containing 1,25-(OH).sub.2 D.sub.3, particularly for psoriasis treatment, are known. See e.g., Morimoto et al., "Topical Administration of 1,25-Dihydroxyvitamin D.sub.3 for Psoriasis: Report of Five Cases", Calcif Tissue Int., Vol. 38, ( 1986), pp. 119-22. See also European Patent Application 512,814 which describes cosmetic compositions containing 1-hydroxycholecalciferol and/or 1,25-(OH).sub.2 D.sub.3. The composition is said to prevent the damaging effects of ultra-violet light on skin and to promote the repair of photodamaged skin.
Vitamin D.sub.3 per se is biologically inactive. Vitamin D.sub.3 when applied topically to skin neither has keratinocyte prodifferentiating activity nor is it converted in the skin to 25-OH-D.sub.3 derivative which is a necessary precursor of 1,25-(OH).sub.2 D.sub.3 metabolite. See MacLaughlin et al., "Cultured Human Keratinocytes Cannot Metabolize Vitamin D.sub.3 to 25-hydroxyvitamin D.sub.3 " Federation of European Biochemical Societies, Vol. 282, No. 2, ;(May 1991), pp. 409-411. Thus, although numerous cosmetic compositions containing vitamin D.sub.3 are known and are available commercially, such compositions do not provide the benefit of 1,25-(OH).sub.2 D.sub.3 induced keratinocyte differentiation.
Unfortunately, topical application of 1,25-(OH).sub.2 D.sub.3 must be carefully controlled, because 1,25-(OH).sub.2 D.sub.3 applied topically increases blood level of 1,25-(OH).sub.2 D.sub.3. The normal concentration of 1,25-(OH).sub.2 D.sub.3 in blood is 10.sup.-12 M. Higher levels of 1,25-(OH).sub.2 D.sub.3 increase blood calcium levels and may cause heart problems, muscle weakness and contraction which may be fatal. When 1,25-(OH).sub.2 D.sub.3 is produced endogenously the problem of toxicity does not occur, because the enzyme that produces 1,25-(OH).sub.2 D.sub.3 from 25-OH-D.sub.3 is shut off when endogenous levels of 1,25-(OH).sub.2 D.sub.3 are at normal circulating levels which is well below toxic levels.
Thus, it is desirable to maintain optimum endogenous production of 1,25-(OH).sub.2 D.sub.3 in skin in order to promote keratinocyte differentiation and, in turn, to maintain or promote a healthy, smooth, young-looking skin. The endogenous production of 1,25-(OH).sub.2 D.sub.3 in the skin is limited, however, by the level of vitamin D.sub.3 produced in the skin and by the blood levels of 25-OH-D.sub.3. Several factors, such as increased skin pigmentation, reduced sunlight, and aging, all decrease the capacity of human skin to produce vitamin D.sub.3. See Holick, M. F. in "Cutaneous Aging" (edited by A. Kligman), pp. 223-246, Univ. of Tokyo Press, Tokyo, 1988.
It is known that exogenously applied 25-OH-D.sub.3 is converted to 1,25-(OH).sub.2 D.sub.3 in keratinocyte cultures. Bikle et al. reported in "Squamous Carcinoma Cell Lines Produce 1,25-Dihydroxyvitamin D, but Fail to Respond to Its Prodifferentiating Effect", The Society for Investigative Dermatology, Inc., (1991), p. 435, that keratinocytes need not depend on exogenously added 1,25-(OH).sub.2 D.sub.3 as they can rapidly convert exogenously added 25-OH-D.sub.3 to 1,25-(OH).sub.2 D.sub.3. It is desirable, however, to maximize the pro-differentiating effect of 25-OH-D.sub.3. Additionally, it is desirable to avoid fast uptake of active ingredients by skin cells in order to attain a prolonged exposure of skin cells to active ingredients. Unfortunately, exogenously added 1,25-(OH).sub.2 D.sub.3 is rapidly degraded within the skin cells. See Bikle et al., "1,25-Dihydroxyvitamin D.sub.3 Production by Human Keratinocytes", The Journal of Clinical Investigation, Inc., Volume 78, (August 1986), pp. 557-566. Slow uptake of active ingredients also minimizes cost of production and maximizes effectiveness of compositions by providing skin cells with steady state levels of ingredients for prolonged periods of time.
The present invention is based at least in part on the discovery that the addition of certain lipids to 25-OH-D.sub.3 results in a synergistic increase in keratinocyte differentiation. The inventive compositions avoid the toxic effects of 1,25-(OH).sub.2 D.sub.3 and increase substantially the prodifferentiating activity of 25-OH-D.sub.3. It has also been found as part of the present invention that the application to skin of 25-OH-D.sub.3 in place of 1,25-(OH).sub.2 D.sub.3 results in slower uptake of the active ingredients by skin cells.
Cosmetic compositions are known which utilize ceramides (lipids found in skin) and pseudoceramides (synthetic molecules resembling ceramides) to control water loss and/or to repair damaged (e.g., dry, flaky, chapped, wrinkled) skin by replacing the skin's natural lipids. See, for example, U.S. Pat. Nos. 5,206,020 (Critchley et al.), 5,198,210 (Critchley et al.), 5,175,321 (Ohashi et al.), 4,985,547 (Yano et al.), 4,778,823 (Kawamata et al.), and European Patent Application 556,957. Ceramides alone do not induce keratinocyte differentiation, except at higher levels. The incentive exists, however, to keep ceramides' level in a formulation at a minimum due to high cost of ceramides.
Okazaki et al. reported in "Role of Ceramide as a Lipid Mediator of 1.alpha.,25-Dihydroxyvitamin D.sub.3 -induced HL-60 Cell Differentiation", The Journal of Biological Chemistry, Vol. 265, No. 26, Sep. 15, 1990. pp. 115823-31, that cell-permeable ceramides with shorter N-acyl chains induce HL-60 cell (human myelocytic leukemia cells) differentiation at subthreshold concentrations of 1,25-(OH).sub.2 D.sub.3. In this regard, it should be noted that while lipids are also included in the inventive compositions, HL-60 cells (pathological tumor cells found in blood) and keratinocytes (normal cells found in skin) are so different from each other in function, their differentiation pathways and biological environment are so diverse and the principles and skills required in formulating cosmetic compositions and anti-tumor compositions are so distinct, that it is difficult to extend the teachings in one of the arts to the other. The fact that some agent induces ,differentiation of HL-60 cells is not necessarily indicative that the same agent will induce differentiation of keratinocytes. For example, retinoic acid induces differentiation of HL-60 cells but prevents differentiation of keratinocytes. Furthermore, in the present invention a lipid ingredient is employed in combination with 25-OH-D.sub.3, not 1,25-(OH).sub.2 D.sub.3. Topical application of 1,25-(OH).sub.2 D.sub.3 is problematic for reasons discussed above.
Accordingly, it is an object of the present invention to provide compositions for treatment of skin, while avoiding the disadvantages of prior art.
It is another object of the present invention to provide a skin treatment composition containing 25-OH-D.sub.3 in combination with an ingredient which enhances the keratinocytes prodifferentiating activity of 25-OH-D.sub.3.
It is yet another object of the invention to provide a skin treatment composition which maximizes the prodifferentiating activity of 1,25-(OH).sub.2 D.sub.3 while avoiding the toxic effects of 1,25-(OH).sub.2 D.sub.3.
It is still another object of the invention to provide a skin treatment composition which is taken up by skin cells slowly.
It is another object of the invention to provide a method for treating or preventing the appearance of wrinkled, flaky, aged, photodamaged skin or skin disorders.
These and other objects of the invention will become more apparent from the detailed description and examples which follow.